PROJECT SUMMARY The human bacterial pathogen Streptococcus pyogenes (group A streptococcus, GAS) causes more than 600 million cases of pharyngitis annually worldwide. The proposed research seeks to discover, on a genome-wide scale, GAS genes required for, or contributing to, pharyngitis, the dominant human disease caused by this pathogen. The pathogen genes and basic molecular pathogenesis processes contributing to the three phases of pharyngitis: initial colonization, acute clinical disease, and persistence in the oropharynx remain poorly understood. This basic science knowledge deficit has severely limited the ability to create new clinical tools such as a successful GAS vaccine. Thus, the studies are designed to address this critical knowledge deficit. We will combine our very recent success in applying transposon-directed insertion site sequencing (TraDIS) technology to GAS with our 18 years of productive experience with a cynomolgus macaque model of pharyngitis, currently the gold standard experimental model for this disease. We will use TraDIS to conduct an in vivo genome-wide screen that will systematically identify GAS genes required for colonization, clinical disease and persistence in the oropharynx of cynomolgus macaques. The goal of the proposed studies is to identify new molecular mechanisms used by GAS to survive in the primate oropharynx. The results of our studies may ultimately help in formulating novel translational strategies to prevent or treat GAS pharyngitis. To achieve our goals, the following two specific aims are proposed: Specific Aim 1: Exploit TraDIS for genome-wide identification of GAS genes required for colonization, acute clinical infection, and persistence (i.e., fitness) in the oropharynx of cynomolgus macaques. Specific Aim 2: Use isogenic gene-knockout mutant strains to validate the importance of two selected candidate genes to GAS pharyngitis identified by the genome-wide screen. The proposed line of research will exploit our innovative and successful application of TraDIS to GAS. It will be the first use of TraDIS in non-human primates and the first study to investigate determinants of GAS virulence in the oropharynx using genome-wide transposon mutagenesis.